ABSTRACT
Bioluminescent reporter genes are sensitive in situ tools for following disease progression in preclinical models, albeit they are subject to scattering and absorption in deep tissues. We have generated a bicistronic Cre/LoxP reporter mouse line that pairs the expression of firefly luciferase with quantifiable expression of a human placental alkaline phosphatase that is secreted into the serum (SeAP). With the use of this dual-modality bioreporter with a novel, inducible Pax7-CreER line for tracking muscle satellite cells, we demonstrate the longitudinal kinetics of muscle stem cell turnover, accounting for a doubling of the signal from satellite cell and progeny every 3.93 wk in the transition from adolescence to early adulthood. We also show that this dual-modality bioreporter can be incorporated in preclinical cancer models, whereby SeAP activity is reflective of tumor burden. Thus, this dual bioreporter permits both spatial localization and accurate quantification of biological processes in vivo even when the tissue of interest is deep within the animal.
Subject(s)
Adult Stem Cells/metabolism , Genes, Reporter , Sarcoma, Experimental/genetics , Sarcoma, Experimental/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Alkaline Phosphatase/genetics , Animals , Base Sequence , DNA Primers/genetics , GPI-Linked Proteins , Humans , Isoenzymes/genetics , Luciferases, Firefly/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , PAX7 Transcription Factor/geneticsABSTRACT
BACKGROUND: Unresectable or metastatic disease represents the greatest obstacle to cure for children with rhabdomyosarcoma. In this study we sought to identify gene expression signatures of advanced stage and progressive disease. PROCEDURE: Using oligonucleotide gene expression analysis for a focused set of 60 genes, we analyzed the myogenic expression profiles of 89 rhabdomyosarcomas from the Intergroup Rhabdomyosarcoma Study-IV. RESULTS: While the expression profile of rhabdomyosarcomas closely paralleled gene expression profiles of normal embryonic myogenic progenitors, growth factors were most closely associated with disease progression. Specifically, we identified platelet-derived growth factor (PDGF) receptors and insulin-like growth factor as strongly correlated with decreased failure-free survival. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) of an independent data set suggested that autocrine growth signaling, if present, is not regulated in a simple manner at the transcriptional level. CONCLUSIONS: Increased transcriptional levels of PDGF receptors and insulin-like growth factor are associated with decreased survival in rhabdomyosarcomas. Dual blockade of these growth-factor-signaling pathways may be a valuable strategy in preclinical therapeutic studies.
Subject(s)
Autocrine Communication , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/biosynthesis , Receptors, Platelet-Derived Growth Factor/biosynthesis , Rhabdomyosarcoma/metabolism , Somatomedins/biosynthesis , Adolescent , Adult , Child , Child, Preschool , Cohort Studies , Disease-Free Survival , Female , Humans , Infant , Male , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Rhabdomyosarcoma/mortality , Transcription, GeneticABSTRACT
Intramembrane cleaving proteases such as site 2 protease, gamma-secretase, and signal peptide peptidase hydrolyze peptide bonds within the transmembrane domain (TMD) of signaling molecules such as SREBP, Notch, and HLA-E, respectively. All three enzymes require a prior cleavage at the juxtamembrane region by another protease. It has been proposed that removing the extracellular domain allows dissociation of substrate TMD, held together by the extracellular domain or loop. Using gamma-secretase as a model intramembrane cleaving protease and Notch as a model substrate, we investigated whether activating and inactivating mutations in Notch modulate gamma-secretase cleavage through changes in oligomerization. We find that although the Notch epidermal growth factor repeats can promote dimer formation, most surface Notch molecules in mammalian cells are monomeric as are constitutively active or inactive Notch1 proteins. Using a bacterial assay for TM dimerization, we find that the isolated TMD of Notch and amyloid precursor protein self-associate and that mutations affecting Notch cleavage by gamma-secretase cleavage do not alter TMD dimerization. Our results indicate that ligand-induced reversal of controlled TMD dimerization by the Notch extracellular domain is unlikely to underlie the regulatory mechanism of intramembranous cleavage.
Subject(s)
Gene Expression Regulation , Membrane Proteins/chemistry , Amino Acid Motifs , Amino Acid Sequence , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases/chemistry , Binding Sites , Biotinylation , Blotting, Western , Cell Line , Cell Membrane/metabolism , Dimerization , Endopeptidases/metabolism , Genes, Reporter , Genetic Vectors , Humans , Immunoprecipitation , Ligands , Mice , Models, Biological , Models, Genetic , Molecular Sequence Data , Mutation , NIH 3T3 Cells , Peptides/chemistry , Plasmids/metabolism , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Receptors, Notch , Sequence Homology, Amino Acid , Signal Transduction , TransfectionABSTRACT
Polymorphisms of glutathione S-transferase (GST) enzymes have been correlated with altered risk of several cancers, as well as altered response and toxicity from cancer chemotherapy. We report a low cost, highly reproducible and specific PCR-based high-throughput assay for genotyping different GSTs designed for use in large clinical trials. In comparison to an alternative genotyping method (single nucleotide extension), the sensitivity and specificity of the high throughput assay was shown to be 92 and 97%, respectively, depending on the source of genomic DNA. Using the high-throughput assay, we demonstrate by multivariate analysis an increased risk of acute lymphoblastic leukemia, glial brain tumors, and osteosarcoma for patients carrying nonnull alleles of GSTM1 and/or GSTT1.
